The invention relates to compositions and methods for the selective inhibition of cytokine-mediated NF-xcexaB activation by blocking the interaction of NEMO with IxcexaB kinase-xcex2 (IKKxcex2) at the NEMO binding domain (NBD). The blockade of IKKxcex2-NEMO interaction results in inhibition of IKKxcex2 kinase activation and subsequent decreased phosphorylation of IxcexaB. Phosphorylation of IxcexaB is an integral step in cytokine-mediated NF-xcexaB activation.
NF-xcexaB is a transcription factor which mediates extracellular signals responsible for induction of genes involved in pro-inflammatory responses (Baltimore et al., (1998) U.S. Pat. No. 5,804,374). NF-xcexaB is anchored in the cytoplasm of most non-stimulated cells by a non-covalent interaction with one of several inhibitory proteins known as IxcexaBs (May and Ghosh, (1997) Semin. Cancer. Biol. 8, 63-73; May and Ghosh, (1998) Immunol. Today 19, 80-88; Ghosh et al., (1998) Annu. Rev. Immunol. 16, 225-260). Cellular stimuli associated with pro-inflammatory responses such as TNFxcex1, activate kinases, which in turn activate NF-xcexaB by phosphorylating IxcexaBs. The kinases that phosphorylate IxcexaBs are called IxcexaB kinases (IKKs).
Phosphorylation targets IxcexaBs for ubiquitination and degradation. The degradation and subsequent dissociation of IxcexaBs from NF-xcexaB reveals the nuclear localization signal on NF-xcexaB, resulting in nuclear translocation of active NF-xcexaB, leading to up-regulation of genes responsive to NF-xcexaB (May and Ghosh, (1997) Semin. Cancer. Biol. 8, 63-73; May and Ghosh, (1998) Immunol. Today 19, 80-88; Ghosh et al., (1998) Annu. Rev. Immunol. 16, 225-260; Siebenlist et al., (1994) Annu. Rev. Cell Biol. 12, 405-455). Phosphorylation of IxcexaBs is therefore an essential step in the regulation of NF-xcexaB mediated pro-inflammatory responses.
The identification and characterization of kinases that phosphorylate IxcexaBs has led to a better understanding of signaling pathways involving NF-xcexaB activation. Several different subtypes of IKK have been identified thus far. IKKxcex1 was initially identified as an IxcexaB kinase induced by TNFxcex1 stimulation in HeLa cells (DiDonato et al., (1997) Nature 388, 548-554). Another IxcexaB kinase homologous to IKKxcex1 was identified, termed IKKxcex2 and determined to be the major IxcexaB kinase induced following TNFxcex1 stimulation (Takeda et al., (1999) Science 284, 313-316; Hu et al., (1999) Science 284, 316-320; Li et al., (1999) Science 284, 321-325; Pot et al., (2000) U.S. Pat. No. 6,030,834; Woronicz and Goeddel (1999) U.S. Pat. No. 5,939,302). IKKxcex1 and IKKxcex2 have an overall homology of 52% and a 65% homology in the kinase domain (Zandi et al., (1997) Cell 91, 243-252).
IxcexaB protein kinases (IKKs) phosphorylate IxcexaBs at specific serine residues. For example, they specifically phosphorylate serines 32 and 36 of IxcexaBxcex1 (Traenckner et al., (1995) EMBO J. 14, 2876-2883; DiDonato et al., (1996) Mol. Cell. Biol. 16, 1295-1304). Phosphorylation of both sites is required to efficiently target IxcexaBxcex1 for degradation. Furthermore, activation of IKKxcex1 and IKKxcex2 is usually in response to NF-xcexaB activating agents and mutant IKKxcex1 and IKKxcex2, which are catalytically inactive, can be used to block NF-xcexaB stimulation by cytokines such as TNFxcex1 and IL-1 (Rxc3xa9gnier et al., (1997) Cell 90, 373-383; Delhase et al., (1999) Science 284, 309-313). IxcexaB protein kinases are therefore essential in the regulation of NF-xcexaB activation processes.
IKKxcex1 and IKKxcex2 have distinct structural motifs including an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper domain. These structural characteristics are unlike other kinases, and the non-catalytic domains are thought to be involved in protein-protein interactions. Proteins which bind to IKKs may therefore be capable of regulating the activity of NF-xcexaB (Marcu et al., (1999) U.S. Pat. No. 5,972,655) and potentially regulating downstream events such as induction of NF-xcexaB. For instance, NEMO (NF-xcexaB Essential Modulator) is a protein which has been identified to bind to IKKs and facilitate kinase activity (Yamaoke et al., (1998) Cell 93, 1231-1240; Rothwarf et al., (1998) Nature 395, 287-300; Mercurio et al., (1999) Mol. Cell. Biol. 19, 1526-1538; Haraj and Sun, (1999) J. Biol. Chem. 274, 22911-22914; Jin and Jeang, (1999) J. Biomed. Sci. 6, 115-120).
Inflammation is defined as the reaction of vascularized living tissue to injury. As such, inflammation is a fundamental, stereotyped complex of cytologic and chemical reactions of affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical or biological agent. Inflammation usually leads to the accumulation of fluid and blood cells at the site of injury, and is usually a healing process. However, inflammation sometimes causes harm, usually through a dysfunction of the normal progress of inflammation. Inflammatory diseases are those pertaining to, characterized by, causing, resulting from, or becoming affected by inflammation. Examples of inflammatory diseases or disorders include, without limitation, asthma, lung inflammation, chronic granulomatous diseases such as tuberculosis, leprosy, sarcoidosis, and silicosis, nephritis, amyloidosis, rheumatoid arthritis, ankylosing spondylitis, chronic bronchitis, scleroderma, lupus, polymyositis, appendicitis, inflammatory bowel disease, ulcers, Sjorgen""s syndrome, Reiter""s syndrome, psoriasis, pelvic inflammatory disease, orbital inflammatory disease, thrombotic disease, and inappropriate allergic responses to environmental stimuli such as poison ivy, pollen, insect stings and certain foods, including atopic dermatitis and contact dermatitis.
Inflammatory diseases present a worldwide problem. Studies of disease burden have re-affirmed that tuberculosis is among the top 10 causes of death in the world. Asthma affects 5% of the adult population and 10-15% of the population of children (Armetti and Nicosia (1999) Boll Chim. Farm. 138(11):599). Asthma is a chronic inflammatory disease that is associated with widespread but variable airflow obstruction.
Sepsis is yet another inflammation disorder and is caused by the presence of various pus-forming and other pathogenic microbes, or their toxins, in the blood or tissues of a subject. Sepsis is characterized by a systemic inflammatory response to bacterial products during infection. The symptoms of sepsis, such as fever, are caused at least in part by the inflammatory response of the body to the infecting agent.
Accordingly, there is still a great need for compounds useful for treating inflammatory disorders.
The present invention provides anti-inflammatory compounds, pharmaceutical compositions thereof, and methods of use thereof for treating inflammatory disorders. The present invention is based, at least in part, on the identification of the NEMO binding domain (NBD) on IxcexaB kinase-xcex1 (IKKxcex1) and on IxcexaB kinase-xcex2 (IKKxcex2).
Accordingly, in one aspect, the present invention provides anti-inflammatory compounds comprising a NEMO binding domain (NBD).
In one embodiment, the present invention provides anti-inflammatory compounds comprising fusions of a NEMO binding domain and at least one membrane translocation domain. In a preferred embodiment, the membrane translocation domain facilitates membrane translocation of the anti-inflammatory compounds of the invention in vivo. The membrane translocation domain may, for example, be the third helix of the antennapedia homeodomain or the HIV-1 Tat protein. In one embodiment, the NEMO binding domain is a polypeptide having the sequence set forth in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.
In another embodiment, the present invention provides anti-inflammatory compounds comprising: (a) peptides which include, or consist of, the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; (b) a fragment of at least three amino acids of the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; (c) peptides which include a conservative amino acid substitution of the amino acid sequences of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; and (d) naturally occurring amino acid sequence variants of the amino acid sequences of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.
In another aspect, this invention provides pharmaceutical compositions comprising the anti-inflammatory compounds of the invention, e.g., pharmaceutical compositions which include one or more pharmaceutically acceptable carriers.
In yet another aspect, the invention features a method of treating an inflammatory disorder, e.g., asthma, lung inflammation or cancer, in a subject. The method includes administering to the subject a therapeutically effective amount of one or more anti-inflammatory compounds of the invention. Without intending to be limited by mechanism, it is believed that the anti-inflammatory compounds of the invention may act (directly or indirectly) by blocking the recruitment of leukocytes into sites of acute and chronic inflammation, by down-regulating the expression of E-selectin on leukocytes, or by blocking osteoclast differentiation.
In another aspect, the present invention provides a method of inhibiting NF-xcexaB-dependent target gene, e.g., E-selectin, expression in a cell. The method includes contacting a cell with an anti-inflammatory compound of the present invention, thereby inhibiting NF-xcexaB-dependent target gene expression in a cell. In yet another aspect, the present invention provides methods of inhibiting NF-xcexaB induction (e.g., IKKxcex1 and/or IKKxcex2 dependent induction) in a cell by contacting a cell with an effective amount of an anti-inflammatory compound of the present invention, thereby inhibiting NF-xcexaB induction in a cell. In one embodiment of this invention, such methods utilize anti-inflammatory compounds which include at least one membrane translocation domain. In still another specific embodiment of this invention, the anti-inflammatory compound s utilized in such methods include amino acid sequences comprising the sequences of SEQ ID NO:2, 4, 5, 6, 11, 12, 16, 17 or 18.
In another aspect, the present invention provides methods of identifying an anti-inflammatory compound. The methods include exposing cells which express NEMO and NF-xcexaB to a test compound; and determining whether the test compound modulates activation of NF-xcexaB by the cell, thereby identifying an anti-inflammatory compound.
In another aspect, the present invention provides methods of identifying an anti-inflammatory compound by exposing cells which express NEMO to a test compound; and determining whether the test compound modulates an activity of NEMO, thereby identifying an anti-inflammatory compound, e.g., a compound which modulates the activity of NEMO.
One particular advantage of the anti-inflammatory compounds of the present invention is that while blocking NF-xcexaB induction via IKK, they do not inhibit the basal activity of NF-xcexaB.
Other features and advantages of the invention will be apparent from the following detailed description and claims.